Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4 Verena Schuettea,1, Maria Embgenbroicha,1, Thomas Ulasa, Meike Welzb, Jonas Schulte-Schreppinga, Astrid M. Draffehna, Thomas Quasta, Katharina Kocha, Melanie Nehringa, Jessica Königa, Annegret Zweynerta, Frederike L. Harmsa, Nancy Steinera, Andreas Limmerb, Irmgard Förstera, Friederike Berberich-Siebeltc, Percy A. Knolled, Dirk Wohlleberd, Waldemar Kolanusa, Marc Beyera, Joachim L. Schultzea, and Sven Burgdorfa,2 aLife and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany; bClinic for Orthopaedics and Trauma Surgery, University Hospital Bonn, 53127 Bonn, Germany; cInstitute of Pathology, University of Wurzburg, 97080 Wurzburg, Germany; and dInstitute of Molecular Immunology, Technische Universität München, 81675 Munich, Germany Edited by Emil R. Unanue, Washington University, St. Louis, MO, and approved July 26, 2016 (received for review April 12, 2016) The mannose receptor (MR) is an endocytic receptor involved in Results serum homeostasis and antigen presentation. Here, we identify + + The Presence of the MR on DCs Reduces Cytotoxic Activity of CD8 T the MR as a direct regulator of CD8 T-cell activity. We demonstrate Cells. To investigate whether the MR influences T-cell activation that MR expression on dendritic cells (DCs) impaired T-cell cytotox- by means distinct from antigen presentation, we made use of + icity in vitro and in vivo. This regulatory effect of the MR was me- Désiré T-cell receptor (DesTCR) transgenic CD8 T cells. Because diated by a direct interaction with CD45 on the T cell, inhibiting its these T cells recognize endogenous proteins (5), the presentation of phosphatase activity, which resulted in up-regulation of cytotoxic such antigens is independent of MR-mediated endocytosis. T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of First, we cultured carboxyfluorescein succinimidyl ester (CFSE)- T-cell tolerance. Inhibition of CD45 prevented expression of B-cell labeled DesTCR T cells on wild-type or MR-deficient DCs and lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound monitored T-cell proliferation. DesTCR T cells activated by MR- the CTLA-4 promoter and regulated its activity. These data demon- deficient DCs proliferated to the same extent as T cells activated by strate that endocytic receptors expressed on DCs contribute to the wild-type DCs (Fig. 1A), indicating that the presence of the MR regulation of T-cell functionality. on the DC does not influence T-cell proliferation. Consistent with these observations, no differences in IL-2 secretion were endocytosis receptors | T-cell activation | tolerance | CD45 observed (Fig. S1A). To analyze whether the presence of the MR influences effector functions of these T cells, we performed an he mannose receptor (MR) is an endocytic receptor be- in vitro cytotoxicity assay. To this end, DesTCR T cells were Tlonging to the C-type lectin family and is expressed by distinct activated by either wild-type or MR-deficient DCs and cocul- populations of dendritic cells (DCs), macrophages, and endo- tured with a mixture of differentially labeled target and control thelial cells (1). It consists of an N-terminal cysteine-rich domain cells. Specific elimination of the target cells was monitored by (CR), a fibronectin type II (FN II) domain, eight C-type lectin- flow cytometry. Interestingly, T cells activated by MR-deficient like domains (CTLDs), a transmembrane region, and a short DCs displayed a markedly increased cytotoxic activity compared intracellular region. The CTLDs of the MR can bind to glyco- with T cells activated by wild-type DCs (Fig. 1B), indicating that conjugates terminated in mannose, fucose, or glucose. Despite the presence of eight CTLDs, only CTLD4 is responsible for Significance carbohydrate binding. Additionally, the MR can bind to sul- fated carbohydrates via its CR and to collagen via its FN II Endocytic receptors regulate the internalization of extracellular domain (1). antigens and are often targeted to induce a potent immune In a previous study, we demonstrated that antigens internalized response (e.g., tumor vaccinations), albeit with limited success. by the MR are processed specifically for cross-presentation (2). Here, we describe a role of an endocytic receptor on the reg- The correlation between the MR and cross-presentation offered ulation of T-cell functionality. We demonstrate that the ex- the possibility that antigens targeted toward the MR could in- pression of the mannose receptor on antigen-presenting cells duce potent cytotoxic T-cell responses. Because the induction of a (APCs) impaired full activation of cytotoxic T cells by direct in- strong cytotoxic T-cell response against tumor-specific antigens is teraction with CD45 on the T-cell surface, resulting in CD45 inhibition, T-cell reprogramming, and the induction of T-cell a crucial process in many tumor vaccination strategies, antigen INFLAMMATION targeting toward the MR seemed to be a promising approach. tolerance. These findings demonstrate that the immune-regu- IMMUNOLOGY AND latory properties of endocytic receptors expressed on APCs However, in in vivo tumor models, antigen targeting toward the have an important impact on the potency of T-cell activation. MR did not result in a strong cytotoxic T-cell response but, instead, led to the induction of antigen-specific T-cell tolerance (3, 4). Author contributions: V.S., M.E., T.Q., A.L., D.W., J.L.S., and S.B. designed research; V.S., Therefore, we analyzed whether the MR has a regulatory effect M.E., M.W., J.S.-S., T.Q., K.K., M.N., J.K., A.Z., F.L.H., and D.W. performed research; N.S., on the induction of immune responses by means distinct from I.F., F.B.-S., P.A.K., W.K., M.B., and J.L.S. contributed new reagents/analytic tools; V.S., M.E., T.U., antigen uptake and presentation, and we investigated a direct M.W., A.M.D., T.Q., D.W., M.B., J.L.S., and S.B. analyzed data; and V.S., M.E., and S.B. wrote the paper. influence of the MR on T-cell activation. We could demonstrate The authors declare no conflict of interest. that the presence of the MR on the antigen-presenting cell (APC) This article is a PNAS Direct Submission. directly impairs the cytotoxic activity of T cells in vitro and Data deposition: The microarray data reported in this paper have been deposited in the in vivo. We showed that this effect was due to a direct interaction Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. of the MR with CD45 on the T-cell surface. This interaction GSE45805). inhibited CD45 activity and resulted in the up-regulation of the 1V.S. and M.E. contributed equally to this work. – inhibitory molecule cytotoxic T-lymphocyte associated Protein 4 2To whom correspondence should be addressed. Email: [email protected]. (CTLA-4), which was responsible for the impaired cytotoxic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activity of the T cells. 1073/pnas.1605885113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1605885113 PNAS | September 20, 2016 | vol. 113 | no. 38 | 10649–10654 Downloaded by guest on September 28, 2021 MR interaction partner between 150 and 230 kDa (Fig. 2A). Be- cause it has been demonstrated before that the CR of the MR bears affinity for the phosphatase CD45 (7), whose molecular mass is within the observed range and is expressed on the surface of + CD8 T cells, we verified whether the observed interaction partner resembles CD45. To this end, we performed coimmunoprecipita- tion experiments with the FcMR protein. Subsequent Western blot Fig. 1. MR expression on DCs reduces cytotoxicity of activated T cells. analysis revealed a clear interaction between CD45 and the MR (A) Proliferation of CFSE-labeled DesTCR T cells cocultured with wild-type or (Fig. 2B). Additionally, we precipitated CD45 from DesTCR T MR-deficient (MR−/−) BM-DCs. (B) Cytotoxic activity of DesTCR T cells acti- cells. Subsequent Western blot analysis after staining with FcMR vated by wild-type (wt) or MR−/− BM-DCs. (C) Cytotoxic activity of DesTCR further confirmed the interaction of the MR with CD45 (Fig. 2C). − − T cells activated by wt or MR / BM-DCs in the presence of 10 μg/mL FcMR or Next, we investigated whether the interaction of the MR with isotype control. Line graphs depict statistical analysis (mean ± SEM) of rep- CD45 affected its phosphatase activity. To this end, we generated licates from at least three independent experiments. cell lysates from DesTCR T cells after incubation with wild-type or MR-deficient DCs. Subsequently, we precipitated CD45 from + the presence of the MR influences the cytotoxic activity of CD8 these lysates and added 4-nitrophenyl phosphate, whose dephos- T cells. Consistent with these findings, restimulation of T cells ini- phorylation can be quantified by colorimetry. Indeed, the phos- tially activated by MR-deficient DCs with a CD3-specific antibody phatase activity of CD45 was reduced when T cells were cocultured resulted in an increased secretion of IFN-γ compared with T cells with MR-bearing DCs (Fig. 2D). Similar results were obtained activated by wild-type DCs (Fig. S1B). Additionally, MR-induced when T cells were cultivated with HEK293T cells that were stably T-cell tolerance was diminished after strong activation of the DC transfected with the MR (8) (Fig. 2E), pointing out an inhibitory with CpG (Fig. S1C), which results in down-regulation of the effect of the MR on CD45 activity. Finally, to demonstrate that ob- MR (Fig. S1 D and E), again demonstrating an inhibitory effect served dephosphorylation indeed was mediated by CD45, we moni- of the MR on T-cell activation.